Catalyst and method for amide formation

ABSTRACT

The invention is a catalyst, the use of the catalyst and a method of using the catalyst in the formation of a compound with an amide bond. The catalyst is an HOBT derivative having the following formula (1):                    
     where R 1  is a group bearing a positive charge at pH 5-7; where Y is a bond or a substituted or unsubstituted alkylene chain containing 1-10 carbon atoms and 0-2 heteroatoms selected from the group consisting of N, S and O; and where X is a linker group selected from —CO— or —SO 2 —. In the method, an amine, a carboxylic acid, an amide coupling agent and a catalyst of formula (1) are reacted for a time sufficient to produce an amide bond between the amine and the carboxylic acid. Thereafter, the compound containing the amide bond is isolated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of pending divisional U.S.Ser. No. 09/633,936, filed Aug. 8, 2000, which is a division of Ser. No.09/213,734, filed Dec. 17, 1998, now U.S. Pat. No. 6,121,471 whichclaims the benefit of Ser. No. 60/068,400, filed Dec. 22, 1997, thecontents of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

This invention provides a novel catalyst and method for the formation ofamide bonds. The catalysts are derivatives of hydroxybenzotriazole(HOBT) and carry a positive charge at pH 5-7. These reagents are usefulfor forming compounds containing an amide bond with high purity andyield.

A wealth of procedures are available for forming amide bonds.Nonetheless, there is still room for improvement. For example,procedures have been developed which use reagents such as carbodiimidesas amide coupling agents. These carbodiimides includedicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).Unfortunately, these particular reagents can require a large excess ofacid, can be problematic for difficult couplings with unreactivecarboxylic acids and/or amines and can be difficult to purify from thereaction mixture.

Catalysts such as 1-hydroxybenzotriazole (HOBT) and derivatives thereofhave also been used.

Unfortunately, HOBT and its derivatives can be difficult to remove fromthe reaction media.

SUMMARY OF THE INVENTION

Briefly stated, the invention is a catalyst, the use of the catalyst anda method of using the catalyst in the formation of a compound with anamide bond. The catalyst is an HOBT derivative having the followingformula (1):

where R¹ is a group bearing a positive charge at pH 5-7; where Y is abond or a substituted or unsubstituted alkylene chain containing 1-10carbon atoms and 0-2 heteroatoms selected from the group consisting ofN, S and O; and where X is a linker group selected from —CO— or —SO₂—.

In accordance with the method aspect of the invention, an amine, acarboxylic acid, an amide coupling agent and a catalyst of formula (1)are reacted for a time sufficient to produce an amide bond between theamine and the carboxylic acid. Thereafter, the compound containing theamide bond is isolated.

In accordance with a preferred method, the reaction is carried out insolution. Also, the amide coupling agent is a carbodiimide, whichcarbodiimide bears a positive charge at pH 5-7. The catalyst is mostpreferably PP-HOBT (see formula (2) below). Finally, the compoundcontaining the amide bond preferably does not bear a positive charge atpH 5-7 and is isolated by passing the entire reaction mixture through acation exchange resin which traps unwanted materials and allows thedesired amide product to be isolated from the eluent with excellentpurity.

This preferred method, wherein the amide coupling agent and catalystboth carry a positive charge at pH 5-7, provides the advantage that thecompound containing the amide bond can be readily isolated from thereaction mixture when the mixture is passed through a cation exchangeresin.

As used herein and the appended claims, the term “catalyst” is intendedto have a relatively broad meaning referring to a compound thatfacilitates the reaction between one or more other compounds, thecatalyst remaining in or returning to its original state.

As used herein and the appended claims, “alkyl” means an unbranched orbranched aliphatic or cyclic group containing 1-20, preferably 1-10,carbon atoms.

As used herein and the appended claims, “alkylene” means an unbranchedor branched carbon chain containing 1-20, preferably 1-10, carbon atoms.Branched carbon chains include both linear and cyclic structures.

As used herein and the appended claims, “aryl” means a mono- orpolycyclic carbocyclic aromatic ring containing 6 to 10 carbon atoms,such as phenyl (Ph) or naphthyl. When more than 1 ring is present, therings can be fused or unfused.

As used herein and the appended claims, “heteroaryl” is a mono-, bi- ortricyclic, N-, O- or S-heteroaryl, such as benzofuran, benzothiophene,furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine,pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline,thiazole, and thiophene.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The catalysts of the present invention have the general formula (1):

where R¹ is a group bearing a positive charge at pH 5-7;

Y is a bond or a substituted or unsubstituted alkylene chain containing1-10 carbon atoms, and, optionally 1 or more heteroatoms such as N, S orO; and

X is a linker group selected from —CO— or —SO₂—.

The function of R¹ is to allow the catalyst of the present invention tobe removed by a cation exchange resin; thereby allowing the formedcompound containing the amide bond to be purified via ion exchangechromatography. R¹ can be any group bearing a positive charge at pH 5-7.R¹ is preferably a substituted or unsubstituted, aliphatic or cyclic,dialkylamine (such that R¹ is a tertiary amine in formula (1)).Preferably, R¹ is a cyclic dialkylamine such as piperidine. Mostpreferably, Y is a bond and R¹ is 4-piperidino-piperidine.

Y is preferably a substituted or unsubstituted alkylene chain containing1-10 carbon atoms.

The substituents for R¹ and Y can be selected for optimization of thesolubility characteristics of the catalyst in various solvents. Forexample, as the number of carbon atoms in the Y substituent increases,the solubility of the catalyst in nonpolar solvents is expected toimprove. Alternatively, groups such as ethers and the like can be addedto the R¹ or Y to enhance solubility in polar solvents.

Suitable substituents for R¹ and Y include groups which are relativelyunreactive with carboxylate groups and amine groups. Examples includeC₁₋₆ alkyl, C₁₋₆ alkoxy, and halogen. Amines and carboxylates are nottypically suitable substituents as this would cause the catalyst toreact with the starting materials, resulting in undesired side products.Presently, —SO₂— is preferred as the linker group X.

The most preferred embodiment of the present invention is a catalyst ofthe formula (2):

This particular catalyst has acceptable solubility in DMF and can bereadily removed from the reaction mixture by passing the mixture througha cation exchange resin.

Preparation of the Catalyst

This most preferred catalyst, which includes a sulphonamide linker groupX, may be synthesized in accordance with the following:

The chemistry depicted in this scheme is derived from Pop et al. (J.Org. Chem. 1997, 62:2594, the disclosure of which is incorporated hereinby reference), who described the preparation of a resin bound HOBT.

When X is a carbonyl group, the catalysts may be prepared from 6-acylchloride-HOBT as shown below:

Method of Amide Bond Formation

The invention provides a method of forming a compound containing anamide bond. The steps comprise reacting an amine, a carboxylic acid, acoupling agent and a compound of the general formula (1) describedabove. The general reaction is represented by the following:

The reaction is carried out under conditions and for a time sufficientto produce an amide bond between the amine and the carboxylic acid.

Suitable amines (R¹) include any compounds containing a free primary orsecondary amine, including, for example, substituted or unsubstitutedalkylamines, dialkylamines, arylamines and heteroarylamines. As iscommonly done, HCl salts or other salts of the amines can also be usedas the starting materials.

Use of the catalyst of the invention provides an advantage when theamine used in the amide formation reaction is provided in the form of anHCl salt. Typically, when using an HCl salt of an amine, a separate baseis added to the reaction mixture to liberate the amine. However, thepresent catalyst, particularly with the tertiary amine in the R¹ group,can fill this role and eliminate the necessity to add a separate base tothe reaction mixture.

Suitable carboxylic acids (R²—CO₂H) include any compounds containing afree carboxylate, including, for example, substituted or unsubstitutedalkylcarboxylates, arylcarboxylates or heteroarylcarboxylates.

The above amines and carboxylates can be substituted 1 or more times(preferably 1 to 3 times) with a halogen, hydroxyl, amine, alkylamine,dialkylamine, alkyl, alkoxy, alkenyl, alkylcarboxylate, thiol, andalkylcarboxyamide. In addition, the starting amines and carboxylic acidsmay have protected functional groups.

In one aspect of this invention, the catalyst of the present inventionis used in the formation of peptide bonds between a first amino acidcontaining moiety and a second amino acid containing moiety. Each ofthese amino acid containing moieties can include one or more aminoacids. Suitable amino acids include naturally encoded amino acidresidues, unusual or unnatural amino acid residues, and synthetic aminoacid residues.

Preferably, the compound containing an amide bond produced by the methodof the present invention is one that does not bear a positive charge atpH 5-7. In this way, the compound can be isolated from the catalyst bypassing the reaction mixture through a cation exchange resin.

Typically, amide coupling agents are compounds that function asdehydrating agents, thus driving the condensation reaction between acarboxylic add and an amine. Preferably, the amide coupling agent is acompound that bears a positive charge at pH 5-7. In this way, the amidecoupling agent may be removed from the reaction solution by the samecation exchange resin as that used for the catalyst. Alternatively, thecoupling agent does not bear a positive charge and other means are usedto remove it from the reaction mixture. For example, precipitation ofthe byproduct of the coupling agent. Moreover, other conventionalchromatographic means may be used to remove the amide coupling agent.

Suitable amide coupling agents includebenzotriazolyloxy-tris(dimethylamino) phosphonium Hexafluorophosphate(BOP) and carbodiimides such as dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC), and1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC). EDC isthe most preferred carbodiimide to use as the amide coupling agent.

Preferably, the reaction is carried out in solution. This isparticularly advantageous because the catalyst, and preferably the amidecoupling agent as well, can be removed from the reaction solution bypassing the solution through a cation exchange resin. Alternatively, thereaction is carried out on solid phase, for example, a solid phasepeptide synthesis such as that described in U.S. Pat. No. 4,888,385, thedisclosure of which is incorporated herein by reference.

Suitable solvents for the reaction include non-nucleophilic organicsolvents, such as dimethylformamide (DMF), dimethylsulfoxide (DMSO),methylene chloride (DCM or CH₂Cl₂) and the like.

The reaction is typically conducted for at least 30 minutes to 24 hours.Preferably, the reaction is conducted for about 12 hours.

The reaction is typically conducted at room temperature (about 25° C.),although any reaction temperature conducive to the reaction can be used.

The compound containing the amide bond can be isolated usingconventional purification means, including, for example, extraction,chromatography, crystallization, etc. However, the preferred method ofisolation involves passing the reaction mixture through a cationexchange resin. Preferred cation exchange resins include silica basedresin substituted with functional groups, including SCX (benzensulfonicacid resin), PRS (propylsulfonic acid resin) and CBA (carboxylic acidresin); all of which are available from Varian Sample Preparation(Harbor City, Calif.). The compound containing the amide bond will washthrough the column, while the catalyst, and preferably the amidecoupling agent as well, will bind to the column. Preferably, about 1 gof resin is used per 0.7 to 1 eq of catalyst.

Using this latter technique the compound containing the amide bond canbe isolated with greater than 95% purity.

The method of the preferred embodiment typically results in greater thanabout 60% yield of compound containing an amide bond. As noted below, ithas been observed that, in comparing otherwise identical amide formationreactions, the presence of the PP-HOBT catalyst made a significantimprovement in the yield over that without its presence.

EXAMPLES

The following examples are provided as illustration only, and are notintended to limit this invention in any way.

Example 1 Procedure for Preparation of PP-HOBT

Starting Materials:

4-chloro-3-nitrobenzenesulfonyl chloride (MW=256) (available fromAldrich) Triethylamine (MW=101)

4-piperidino-piperidine (MW=168) (available from Aldrich, 90%, Lot#11033PG) Hydrazine hydrate

Procedure:

To a stirred solution of 7.68 g (30 mmol) sulfonyl chloride in 120 ml ofdichloromethane was added dropwise, over a 10 min period, 5.049 (30mmol) of 4-piperidino-piperidine and 3.6 g (36 mmol) of triethylamine in30 ml of dichloromethane. A mildly exothermic reaction ensued. Afterstirring 2 hours at room temperature the orange solution was dilutedwith 100 ml of dichloromethane and washed with 2, 100-ml portions of 10%sodium bicarbonate solution and 100 ml of brine. After drying (sodiumsulfate) solvent was removed at reduced pressure to afford 10.7 g ofcrude product as a light tan solid (R_(f)=0.5, Silica, 10%MeOH/chloroform).

To this crude material was added 200 ml of 95% EtOH/5% MeOH followed by60 ml of hydrazine hydrate. The mixture-was refluxed for 3 hours. Overthe first ½ hour the initially orange solution turned deep red-orangebefore turning orange again. At the end of the three hour reflux periodmost of the solvent, water and hydrazine was removed at reducedpressure. To the residue was added 50 ml of EtOH and solvent removed atreduced pressure. This was repeated 2 or more times to give a tan solidwhich was further dried in the vacuum oven to a constant weight of 13.5g.

To the flask containing this solid was added 250 ml of water. Almost allof the solid went into solution, then a fine light yellow precipitateformed. After stirring, the mixture was cooled in an ice bath for twohours. The solid was collected by vacuum filtration through a sinteredglass filter, and rinsed with about 20 ml of cold water. Drying in thevacuum oven at 40° overnight afforded 7.3 g of product as an off-whitecrunchy powder, mp 195-200 (dec). The elemental analysis was consistentwith the compound existing as a monohydrate. With a production of 7.3 g(19 mmol) the yield was calculated as 63%.

An analog of PP-HOBT was prepared using the same procedure butsubstituting 3-dimethylamino propylamine for the4-piperidino-piperidine. This material was poorly soluble in DMF anddichloromethane.

Example 2 Procedure for Amide Formation Using SCX to Remove ExcessAmine, EDC, Urea and the PP-HOBT Catalyst

Starting Materials:

2,6-dimethyl benzoic acid (MW=150)

4-methoxy benzyl amine (MW=137)

EDC HCl salt (MW=191.7)

PP-HOBT (MW=365)

DMF, CH₂Cl₂

Procedure:

The following stock solutions were prepared:

0.1 M acid in 10% DMF/DCM

0.35 M amine in 10% DMF/DCM

0.175 M PP-HOBT in DMF (heat to get into solution)

0.1 M EDC HCl salt in DCM

In a 3 ml vial was added 0.3 ml (30 umol, 1 eq) of the acid stocksolution followed by 0.1 ml (35 umol, 1.16 eq) amine stock solution, 0.2ml (35 umol, 1.16 eq) PP-HOBT stock solution and, finally, 0.4 ml (40umol, 1.33 eq) EDC solution. The capped vial was gently rotated for 12hours, uncapped, and the heterogeneous reaction mixture diluted with 0.5ml of MeOH. This mixture was applied to a 500 mg SCX column pre-washedwith 2.5 ml of MeOH and 2.5 ml of 10% MeOH/CHCl₃. After pushing throughthe solution with nitrogen, the column was washed with 2.5 ml of 10%MeOH/CHCl₃. Evaporation of solvent, first under nitrogen on the hotplate and then in the vacuum oven, afforded 7.5 mg (28 umol, 93% yield)of product which was pure by NMR.

Examples 3 and 4 Comparison of Amide Formation with and without PP-HOBT

To verify the ability of PP-HOBT to catalyze amide formation thefollowing experiments were performed:

Example 3 Amide Formation With PP-HOBT

In a 3 ml vial was added 0.3 ml (30 umol, 1 eq) of the acid stocksolution followed by 0.1 ml (35 umol, 1.16 eq) amine stock solution, 0.2ml (35 umol, 1.16 eq) PP-HOBT stock solution and, finally, 0.4 ml (40umol, 1.33 eq) EDC solution. The capped vial was gently rotated for 2hours, uncapped, and the heterogeneous reaction mixture diluted with 0.5ml of MeOH. This mixture was applied to a 500 mg SCX column pre-washedwith 2.5 ml of MeOH and 2.5 ml of 10% MeOH/CHCl₃. After pushing throughthe solution with nitrogen, the column was washed with 2.5 ml of 10%MeOH/CHCl₃. Evaporation of solvent, first under nitrogen on the hotplate and then in the vacuum oven, afforded 7.5 mg of crude productwhich contained 70% desired amide product and 30% starting acid by NMR.

Example 4 Amide Formation Without PP-HOBT

Example 4 was done under identical conditions to 3 except that noPP-HOBT was added to the reaction mixture. Evaporation of solvent, firstunder nitrogen on the hot plate and then in the vacuum oven, afforded4.0 mg of crude product which contained <5% desired amide product and95% starting acid by NMR.

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed is:
 1. A method of forming and isolating a compoundcontaining an amide bond, comprising the steps of: forming a reactionmixture comprising an amine, a carboxylic acid, a carbodimide agent anda compound of the formula (2):

reacting said reaction mixture under conditions and for a timesufficient to produce an amide bond between the amine and the carboxylicacid; and isolating the compound containing the amide bond by passingthe entire reaction mixture through a cation exchange resin.